The present invention relates to recombinant proteins obtained from the combination of structural domains derived from the a subunits of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP).
In particular, the engineered factors of the invention are obtained by combination of the hairpin loop and kringle domains of the xcex1 chains of HGF and/or MSP, so as to obtain a structure having two superdomains with an intervening linker sequence. Moreover, the invention relates to DNA sequences encoding the above mentioned recombinant proteins, to the expression vectors comprising said DNA sequences and to host cells containing said expression vectors. The recombinant proteins of the present invention are biologically active, and their activity can be measured by determination of their ability to induce activation of the Met tyrosine kinase receptor, their xe2x80x9cscatteringxe2x80x9d effect on epithelial cells, and their protective effect against cell death induced by chemotherapic drugs (vide infra). Therefore, these molecules can conveniently be used to prevent or treat the toxic side effects of the chemotherapeutical treatment of tumours, and to reduce iatrogenic cell damage induced by other types of drugs.
Hepatocyte Growth Factor (HGF) and Macrophage Stimulating Protein (MSP) are highly related proteins both structurally and functionally (FIGS. 1 and 2). Both these factors are secreted as an inactive precursor, which is processed by specific proteases which recognise a cleavage site inside the molecule, dividing the protein in two subunits. These subunits, named xcex1 chain and xcex2 chain, are linked by a disulphide bond. Thus, the mature factor is an xcex1-xcex2 dimeric protein. Only the mature (dimeric) form of the factor is able to activate its receptor at the surface of the target cells (the Met tyrosine kinase in the case of HGF and the Ron tyrosine kinase in the case of MSP) and therefore to mediate biological responses (Naldini, L. et al., 1992, EMBO J. 11: 4825-4833; Wang, M. et al., 1994, J. Biol. Chem. 269; 3436-3440; Bottaro, D. et al., 1991, Science 25: 802-804; Naldini, L. et al., 1991, EMBO J. 10: 2867-2878; Wang, M. et al., 1994, Science 266: 117-119; Gaudino, G. et al., 1994, EMBO J. 13: 3524-3532).
The xcex1 chain of both factors contains a hairpin loop (HL) structure and four domains with a tangle-like structure named kringles (K1-K4; Nakamura T et al., 1989, Nature 342:440-443; Han, S. et al., 1991, Biochemistry 30: 9768-9780). The precursor also contains a signal sequence (LS) of 31 amino acids (in the case of HGF) or of 18 amino acids (in the case of MSP), removed in rough endoplasmic reticulum, which directs the neoformed peptide to the secretive pathway. The xcex2 chain contains a box with a sequence homologous to that typical of serine proteases, but it has no catalytic activity (Nakamura T et al., 1989, Nature 342:440-443; Han, S. et al., 1991, Biochemistry 30: 9768-9780). Both xcex1 and xcex2 chains contribute to the binding of the growth factor to the respective receptor (Met for HGF and Ron for MSP).
HGF and MSP polypeptides are able to induce a variety of biological effects besides cell proliferation. The main biological activities of these molecules are: stimulation of cell division (mitogenesis); stimulation of motility (scattering); induction of polarisation and cell differentiation; induction of tubule formation (branched morphogenesis); increase of cell survival (protection from apoptosis). The tissues that respond to HGF and MSP stimulation are those where cells express the respective Met (HGF) and Ron (MSP) receptors. The most important target tissues of these factors are epithelial cells of different organs, such as liver, kidney, lung, breast, pancreas and stomach, and some cells of the hematopoietic and nervous systems. A detailed review of the biological effects of HGF and MSP in the various tissues can be found in Tamagnone, L. and Comoglio, P., 1997, Cytokine and Growth Factor Re-views, 8: 129-142, Elsevier Science Ltd.; Zarnegar, R. and Michalopoulos, G., 1995, J. Cell Biol. 129: 1177-1180; Medico, E. et al., 1996, Mol. Biol. Cell, 7: 495-504; Banu, N. et al., 1996, J. Immunol. 156: S2933-2940.
In the case of HGF, the hairpin loop and the first two kringles are known to contain the sites of direct interaction with the Met receptor (Lokker NA et al., 1992, EMBO J., 11:2503-2510; Lokker, N. et al., 1994, Protein Engineering 7: 895-903). Two naturally-occurring truncated forms of HGF produced by some cells by alternative splicing have been described. The first one comprises the first kringle (NKI-HGF Cioce, V. et al., 1996, J. Biol. Chem. 271: 13110-13115) whereas the second one spans to the second kringle (NK2-HGF Miyazawa, K. et al., 1991, Eur. J. Biochem. 197: 15-22). NK2-HGF induces cell scattering, but it is not mitogenic as the complete growth factor is (Hartmann, G. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11574-11578). However, NK2-HGF regains mitogenic activity in the presence of heparin, a glucosaminoglycan that binds HGF through a domain contained in the first kringle and which is likely to induce dimerization of NK2-HGF (Schwall, R. et al., 1996, J. Cell Biol. 133: 709-718). Moreover NK2-HGF, being a partial agonist of Met, behaves as a competitive inhibitor of HGF as far as the mitogenic activity is concerned (Chan, A. et al., 1991, Science 254: 1382-1385). NK1-HGF has also been described to exert partial stimulation of Met and competitive inhibition of HGF mitogenic activity (Cioce, V. et al., 1996, J. Biol. Chem. 27: 13110-13115). Anyway, a truncated factor is endowed with an activity markedly lower than the recombinant factors described in the invention, as shown in example 3.
In the case of MSP, the interaction sites with the Ron receptor are less understood: some preliminary studies suggest a situation opposite of that of HGF, i.e. the xcex2 chain directly binds the receptor whereas the xcex1 chain would act stabilizing the complex (Wang MH et al., 1997, J. Biol. Chem. 272:16999-17004).
The therapeutical use of molecules such as HGF and MSP is potentially valuable in a wide range of pathologies (Abdulla, S., 1997, Mol. Med. Today 3: 233). Nevertheless, a number of technical as well as biological complications make the application of these molecules in clinics difficult. First of all, the pleiotropic character of these factors can causes poorly selective biological responses, which involve undesired side effects. For example, the use of HGF to prevent some side effects of the chemotherapeutic drug cisplatin has been proposed (Kawaida K et al., 1994, Proc. Natl. Acad. Sci. 91:4357-4361). Cancer patients treated with this drug can suffer kidney acute damage due to the cytotoxic action of cisplatin on proximal tubule epithelial cells. HGF is able to protect these cells against programmed death (apoptosis) induced by cisplatin, but at the same time it can induce an undesired proliferation of neoplastic cells. Other problems related to the pharmaceutical use of HGF and MSP are the necessity of their proteolytic activation and their stability, which causes technical problems. The NK1 and NK2 truncated forms of HGF do not require proteolytic activation, but they have a reduced biological activity.
The present invention provides recombinant molecules composed of a combination of structural domains derived from the xcex1 chains of HGF and/or MSP, which overcome the problems of the prior art molecules described above. The molecules of this invention are composed of two superdomains connected by a linker. Each superdomain is composed of a combination of the HL and K1-K4 domains of the xcex1 chain of HGF and/or MSP. These engineered factors induce selective biological responses, do not require proteolytic activation, are stable and are more active than the truncated forms of HGF described previously.
The present invention relates to recombinant proteins (which will be hereinafter referred to indifferently as proteins, molecules, engineered or recombinant factors) characterised by a structure that comprises two superdomains, each consisting of a combination of HL and K1-K4 domains derived from the xcex1 chain of HGF and/or MSP, linked by a spacer sequence or a linker. In particular, the invention relates to proteins of general formula (I)
[A]xe2x88x92Bxe2x88x92[C]xe2x88x92(D)yxe2x80x83xe2x80x83(I)
in which
[A] corresponds to the sequence (LS)m-HL-K1-(K2)n-(K3)o-(K4)p wherein (the numbering of the following amino acids refers to the HGF and MSP sequences as reported in FIGS. 1 and 2, respectively):
LS is an amino acid sequence corresponding to residues 1-31 of HGF or 1-18 of MSP;
HL is an amino acid sequence derived from the xcex1 chain of HGF starting between residues 32-70 and ending between residues 96-127; or it is an amino acid sequence derived from the xcex1 chain of MSP starting between residues 19-56 and ending between residues 78-109;
K1 is an amino acid sequence derived from the xcex1 chain of HGF starting between residues 97-128 and ending between residues 201-205; or it is an amino acid sequence derived from the xcex1 chain of MSP starting between residues 79-110 and ending between residues 186-190;
K2 is an amino acid sequence derived from the xcex1 chain of HGF starting between residues 202-206 and ending between residues 283-299; or it is an amino acid sequence derived from the xcex1 chain of MSP starting between residues 187-191 and ending between residues 268-282;
K3 is an amino acid sequence derived from the xcex1 chain of HGF starting between residues 284-300 and ending between residues 378-385; or it is an amino acid sequence derived from the xcex1 chain of MSP starting between residues 269-283 and ending between residues 361-369;
K4 is an amino acid sequence derived from the xcex1 chain of HGF starting between residues 379-386 and ending between residues 464-487; or it is an amino acid sequence derived from the xcex1 chain of MSP starting between residues 362-370 and ending between residues 448-481;
m, n, o, p can be 0 or 1;
the sum n+o+p is an integer from 1 to 3 or 0, with the proviso that nxe2x89xa7oxe2x89xa7p;
B is the sequence [(X)qY]r, wherein X=Gly and Y=Ser, or Cys, or Met, or Ala;
q is an integer from 2 to 8;
r is an integer from 1 to 9;
[C] corresponds to the sequence HL-K1-(K2)s-(K3)t-(K4)u wherein HL, K1-K4 are as defined above,
s, t, u are 0 or 1; the sum s+t+u is an integer from 1 to 3 or 0, with the proviso that sxe2x89xa7txe2x89xa7u;
D is the sequence W-Z, wherein W is a conventional proteolytic site, Z is any tag sequence useful for the purification and detection of the protein; y is 0 or 1.
Non-limiting examples of W are consensus sequences for enterokinase protease, thrombin, factor Xa and IgA protease.
Preferred proteins of general formula (I), are those in which: the HL domain is a sequence of HGF xcex1 chain ranging from amino acids 32 to 127, or a sequence of MPS xcex1 chain ranging from amino acids 19 to 98; the K1 domain is a sequence of HGF xcex1 chain ranging from amino acids 128 to 203, or a sequence of MPS xcex1 chain ranging from amino acids 99 to 188; the K2 domain is a sequence of HGF xcex1 chain ranging from amino acids 204 to 294, or a sequence of MPS xcex1 chain ranging from amino acids 189 to 274; the K3 domain is a sequence of HGF xcex1 chain ranging from amino acids 286 to 383, or a sequence of MPS xcex1 chain ranging from amino acids 275 and 367; the K4 domain is a sequence of HGF xcex1 chain ranging from amino acids 384 to 487, or a sequence of MPS xcex1 chain ranging from amino acids 368 and 477.
Among the possible combinations of the domains of general formula (I), the following (II) and (III) are preferred, concerning two recombinant factors named Metron Factor-1 and Magic Factor-1, respectively:
LSMSP-HLMSP-K1MSP-K2MSP
-L-HLHGF-K1HGF-K2HGF-D 
(Metron Factor-1)xe2x80x83xe2x80x83(II)
and
LSHGF-HLHGF-K1HGF-K2HGP
-L-HLHGF-K1HGF-K2HGF-D 
(Magic Factor-1)xe2x80x83xe2x80x83(III)
For both molecules, L is a linker sequence (Gly4Ser)3, D is a tag sequence Asp4-Lys-His6.
For Metron Factor-1, LSMSP is the sequence 1-18 of MSP, HLMSP is the sequence 19-56 of MSP, K1MSP is the sequence 99-188 of MSP, K2MSP is the sequence 189-274 of MSP, HLHGF is the sequence 32-127 of HGF, K1HGF is the sequence 128-203 of HGF, K2HGF is the sequence 204-294 of HGF.
For Magic Factor-1, HLHGF, K1HGF, K2HGF are as defined above, LSHGF is the sequence 1-31 of HGF.
The hybrid molecules of the invention are prepared by genetic engineering techniques according to a strategy involving the following steps:
a) construction of DNA encoding the desired protein;
b) insertion of DNA in an expression vector;
c) transformation of a host cell with recombinant DNA (rDNA);
d) culture of the transformed host cell so as to express the recombinant protein;
e) extraction and purification of the produced recombinant protein.
The DNA sequences corresponding to HGF or MSP structural domains can be obtained by synthesis or starting from DNA encoding for the two natural factors. For example, screening of cDNA libraries can be carried out using suitable probes, so as to isolate HGF or MSP cDNA. Alternatively, HGF or MSP cDNA can be obtained by reverse transcription from purified mRNA from suitable cells.
cDNAs coding for the fragments of HGF and MSP xcex2 chains can be amplificated by PCR (Mullis, K. B. and Faloona, F. A., 1987, Methods in Enzymol. 155, 335-350), and the amplification products can be recombined making use of suitable restriction sites, naturally occurring in the factor sequences or artificially introduced in the oligonucleotide sequence used for the amplification.
In greater detail, one of the above mentioned strategies can be the following:
the portions of DNA encoding the LS, HL, K1, K2, K3 and K4 domains are amplificated by PCR from HGF or MSP cDNA and then recombined to obtain the hybrid sequences corresponding to [A] and [C]. Oligonucleotides recognising sequences located at the two ends of the domains to be amplificated are used as primers. Primers are designed so as to contain a sequence allowing recombination between the DNA of a domain and the adjacent one. Said recombination can be carried out by endonuclease cleavage and subsequent ligase reaction, or making use of the recombinant PCR method (Innis, N A et al., 1990, in PCR Protocols, Academic Press, 177-183).
The sequence encoding the domain B (linker) can be obtained by synthesis of a double chain oligonucleotide, which can be inserted between [A] and [C] using suitable restriction sites.
The resulting three fragments encoding for [A], [B] and [C] are then inserted in the correct sequence in a suitable vector. In this step it can be decided whether to add or not the domain D (tag), obtained by synthesis analogously to domain B, downstream fragment [C].
The recombinant expression vector can contain, in addition to the recombinant construct, a promoter, a ribosome binding site, an initiation codon, a stop codon, optionally a consensus site for expression enhancers.
The vector can also comprise a selection marker for isolating the host cells containing the DNA construct. Yeast or bacteria plasmids, such as plasmids suitable for Escherichia Coli, can be used as vectors, as well as bacteriophages, viruses, retroviruses, or DNA.
The vectors are cloned preferably in bacterial cells, for example in Escherichia Coli, as described in Sambrook J., 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, and the colonies can be selected, for example, by hybridisation with radiolabelled oligonucleotide probes; subsequently, the rDNA sequence extracted from the positive colonies is determined by known methods.
The vector with the recombinant construct can be introduced in the host cell according to the competent cell method, the protoplast method, the calcium phosphate method, the DEAE-dextran method, the electric impulses method, the in vitro packaging method, the viral vector method, the micro-injection method, or other suitable techniques.
Host cells can be prokaryotic or eukaryotic, such as bacteria, yeasts or mammal cells, and they will be such as to effectively produce the recombinant protein.
After transformation, cells are grown in a suitable medium, which can be for example MEM, DMEM or RPMI 1640 in the case of mammal host cells.
The recombinant protein is secreted in the culture medium from which it can be recovered and purified with different methods, such as mass exclusion, absorption, affinity chromatography, salting-out, precipitation, dialysis, ultrafiltration.
A simple, rapid system for the production of the molecules of the invention is, for example, transient-expression in mammal cells.
Accordingly, the plasmid containing the recombinant DNA fragment, for example PMT2 (Sambrook, J. et al., 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press), is transfected in suitable recipient cells, such as Cos7 (Sambrook, J. et al., supra) by the calcium phosphate technique or other equivalent techniques. Some days after transfection, the conditioned medium of the transfected cells is collected, cleared by centrifugation and analysed for its content in factor. For this analysis, antibodies directed against HGF or MSP, or against any tag sequence, can be used: the supernatant is immunoprecipitated and then analysed by western blot with the same antibody. The supernatant containing the recombinant factor can also be used directly for biochemical and biological tests. The protein can be purified, for example, using a poly-histidine tag sequence, by absorption on a nickel resin column and subsequent elution with imidazole.
The biochemical properties. of the recombinant factors of the invention were tested in connection with their ability to activate Met and Ron receptors.
Sub-micromolar concentrations of the factors have proved to induce phosphorylation in Met tyrosine in human epithelial cells A549, whereas they do not induce phosphorylation above basal values in cells expressing Ron. On the whole, the tests proved that the first two kringles of HGF maintain their ability to interact and to activate Met tyrosine kinase receptor, whereas the corresponding first two kringles of MSP are not sufficient for modulating the catalytic activity of the Ron receptor. However, the interaction with Ron, although at low affinity, can contribute to the recruitment of the factor at the cell surface, playing a similar role to low affinity receptors (of mature glycoprotein) which recruit the HGF intact molecule through the heparin-binding domain.
The molecules of the invention have a marked biological activity, measured by the scattering tests, and a protecting activity against cell apoptosis induced by cisplatin or etoposide.
In particular, the supernatant containing the recombinant factor has been found to promote scattering of epithelial cells of various nature even at nanomolar concentrations. In these tests, kidney epithelial cells (MDCK) or hepatocyte precursors (MLP29) were used.
In an in vitro experimental system, in which DNA fragmentation typical of apoptotic cells is evaluated by the TUNEL method (Gavrieli, Y. et al., 1992, J. Cell. Biol. 117, 493-501), the recombinant factors protect against apoptosis induced by chemotherapeutic drugs at levels comparable with HGF and remarkably higher than MSP. The engineered molecules proved to be active on human primary epithelial cells from proximal tubule (PTECs), on an immortalised PTECs line (Loc) and on the already cited murine hepatocytes MLP29.
Among the applications of the recombinant molecules of the invention, the following can be cited:
prevention of myelotoxicity; in particular they can be used for the expansion of marrow precursors, to increase proliferation of the hematopoietic precursors or to stimulate their entry in circle;
prevention of liver and kidney toxicity, and of mucositis following antineoplastic treatments; in particular the recombinant factors can be used to prevent toxicity (apoptosis) on differentiated cell elements of liver, kidney and mucosa of the gastroenteral tract, and to stimulate staminal elements of cutis and mucosas to allow the regeneration of germinative layers;
prevention of chemotherapeutic neurotoxicity.
In general, the proteins of the invention provide the following advantages, compared with the parent molecules HGF and MSP: they are smaller molecules with a more compact structure;
they are more stable and are produced in higher amounts;
they require no endoproteolytic cleavage for activation, which transforms the HGF and MSP precursors into the respective active forms;
they can be engineered in combinations of different functional domains, thereby modulating the biological effects, increasing the favourable ones and reducing those undesired (for example, protection from apoptosis versus cell proliferation).
The invention has to be considered also directed at amino acid and nucleotide sequences referred to formula (I), having modifications which can, for example, derive from degeneration of genetic code, without therefore modifying the amino acid sequence, or from the deletion, substitution, insertion, inversion or addition of nucleotides and/or bases according to all the possible methods known in the art.
Furthermore, the invention relates to the expression vectors comprising a sequence encoding for a protein of general formula (I), which can be plasmids, bacteriophages, vituses, retroviruses, or others, and to host cells containing said expression vectors,
Finally, the invention relates to the use of the recombinant proteins as therapeutical agents, and to pharmaceutical compositions containing an effective amount of the recombinant proteins together with pharmacologically acceptable excipients.